Inbred corn line HX894

ABSTRACT

An inbred corn line, designated HX894, is disclosed. The invention relates to the seeds of inbred corn line HX894, to the plants of inbred corn line HX894 and to methods for producing a corn plant, either inbred or hybrid, by crossing the inbred line HX894 with itself or another corn line. The invention further relates to methods for producing a corn plant containing in its genetic material one or more transgenes and to the transgenic plants produced by that method and to methods for producing other inbred corn lines derived from the inbred HX894.

BACKGROUND OF THE INVENTION

[0001] The present invention relates to a new and distinctive corninbred line, designated HX894. There are numerous steps in thedevelopment of any novel, desirable plant germplasm. Plant breedingbegins with the analysis and definition of problems and weaknesses ofthe current germplasm, the establishment of program goals, and thedefinition of specific breeding objectives. The next step is selectionof germplasm that possess the traits to meet the program goals. The goalis to combine in a single variety or hybrid an improved combination ofdesirable traits from the parental germplasm. These important traits mayinclude higher yield, resistance to diseases and insects, better stalksand roots, tolerance to drought and heat, and better agronomic quality.

[0002] Choice of breeding or selection methods depends on the mode ofplant reproduction, the heritability of the trait(s) being improved, andthe type of cultivar used commercially (e.g., F₁ hybrid cultivar,pureline cultivar, etc.). For highly heritable traits, a choice ofsuperior individual plants evaluated at a single location will beeffective, whereas for traits with low heritability, selection should bebased on mean values obtained from replicated evaluations of families ofrelated plants. Popular selection methods commonly include pedigreeselection, modified pedigree selection, mass selection, and recurrentselection.

[0003] The complexity of inheritance influences choice of the breedingmethod. Backcross breeding is used to transfer one or a few favorablegenes for a highly heritable trait into a desirable cultivar. Thisapproach has been used extensively for breeding disease-resistantcultivars. Various recurrent selection techniques are used to improvequantitatively inherited traits controlled by numerous genes. The use ofrecurrent selection in self-pollinating crops depends on the ease ofpollination, the frequency of successful hybrids from each pollination,and the number of hybrid offspring from each successful cross.

[0004] Each breeding program should include a periodic, objectiveevaluation of the efficiency of the breeding procedure. Evaluationcriteria vary depending on the goal and objectives, but should includegain from selection per year based on comparisons to an appropriatestandard, overall value of the advanced breeding lines, and number ofsuccessful cultivars produced per unit of input (e.g., per year, perdollar expended, etc.).

[0005] Promising advanced breeding lines are thoroughly tested andcompared to appropriate standards in environments representative of thecommercial target area(s) for three years at least. The best lines arecandidates for new commercial cultivars; those still deficient in a fewtraits are used as parents to produce new populations for furtherselection.

[0006] These processes, which lead to the final step of marketing anddistribution, usually take from eight to 12 years from the time thefirst cross is made. Therefore, development of new cultivars is atime-consuming process that requires precise forward planning, efficientuse of resources, and a minimum of changes in direction.

[0007] A most difficult task is the identification of individuals thatare genetically superior, because for most traits the true genotypicvalue is masked by other confounding plant traits or environmentalfactors. One method of identifying a superior plant is to observe itsperformance relative to other experimental plants and to a widely grownstandard cultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

[0008] The goal of plant breeding is to develop new, unique and superiorcorn inbred lines and hybrids. The breeder initially selects and crossestwo or more parental lines, followed by repeated selfing and selection,producing many new genetic combinations. The breeder can theoreticallygenerate billions of different genetic combinations via crossing,selfing and mutations. The breeder has no direct control at the cellularlevel. Therefore, two breeders will never develop the same line, or evenvery similar lines, having the same corn traits.

[0009] Each year, the plant breeder selects the germplasm to advance tothe next generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions, and further selections arethen made, during and at the end of the growing season. The inbred lineswhich are developed are unpredictable. This unpredictability is becausethe breeder's selection occurs in unique environments, with no controlat the DNA level (using conventional breeding procedures), and withmillions of different possible genetic combinations being generated. Abreeder of ordinary skill in the art cannot predict the final resultinglines he develops, except possibly in a very gross and general fashion.The same breeder cannot produce the same line twice by using the exactsame original parents and the same selection techniques. Thisunpredictability results in the expenditure of large research monies todevelop a superior new corn inbred line.

[0010] The development of commercial corn hybrids requires thedevelopment of homozygous inbred lines, the crossing of these lines, andthe evaluation of the crosses. Pedigree breeding and recurrent selectionbreeding methods are used to develop inbred lines from breedingpopulations. Breeding programs combine desirable traits from two or moreinbred lines or various broad-based sources into breeding pools fromwhich inbred lines are developed by selfing and selection of desiredphenotypes. The new inbreds are crossed with other inbred lines and thehybrids from these crosses are evaluated to determine which havecommercial potential.

[0011] Pedigree breeding is used commonly for the improvement ofself-pollinating crops or inbred lines of cross-pollinating crops. Twoparents which possess favorable, complementary traits are crossed toproduce an F₁. An F₂ population is produced by selfing one or severalF₁'s or by intercrossing two F₁'s (sib mating). Selection of the bestindividuals is usually begun in the F₂ population; then, beginning inthe F₃, the best individuals in the best families are selected.Replicated testing of families, or hybrid combinations involvingindividuals of these families, often follows in the F₄ generation toimprove the effectiveness of selection for traits with low heritability.At an advanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease as new cultivars.

[0012] Mass and recurrent selections can be used to improve populationsof either self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

[0013] Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line which is the recurrent parent. The source of the trait tobe transferred is called the donor parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype of the donor parentare selected and repeatedly crossed (backcrossed) to the recurrentparent. The resulting plant is expected to have the attributes of therecurrent parent (e.g., cultivar) and the desirable trait transferredfrom the donor parent.

[0014] Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Allard, 1960; Simmonds, 1979; Sneep et al., 1979; Fehr,1987).

[0015] Proper testing should detect any major faults and establish thelevel of superiority or improvement over current cultivars. In additionto showing superior performance, there must be a demand for a newcultivar that is compatible with industry standards or which creates anew market. The introduction of a new cultivar will incur additionalcosts to the seed producer, the grower, processor and consumer; forspecial advertising and marketing, altered seed and commercialproduction practices, and new product utilization. The testing precedingrelease of a new cultivar should take into consideration research anddevelopment costs as well as technical superiority of the finalcultivar. For seed-propagated cultivars, it must be feasible to produceseed easily and economically.

[0016] Once the inbreds that give the best hybrid performance have beenidentified, the hybrid seed can be reproduced indefinitely as long asthe homogeneity of the inbred parent is maintained. A single-crosshybrid is produced when two inbred lines are crossed to produce the F₁progeny. A double-cross hybrid is produced from four inbred linescrossed in pairs (A×B and C×D) and then the two F₁ hybrids are crossedagain (A×B)×(C×D). Much of the hybrid vigor exhibited by F₁ hybrids islost in the next generation (F₂). Consequently, seed from hybridvarieties is not used for planting stock.

[0017] Hybrid corn seed is typically produced by a male sterility systemincorporating manual or mechanical detasseling. Alternate strips of twocorn inbreds are planted in a field, and the pollen-bearing tassels areremoved from one of the inbreds (female). Providing that there issufficient isolation from sources of foreign corn pollen, the ears ofthe detasseled inbred will be fertilized only from the other inbred(male), and the resulting seed is therefore hybrid and will form hybridplants.

[0018] The laborious, and occasionally unreliable, detasseling processcan be avoided by using cytoplasmic male-sterile (CMS) inbreds. Plantsof a CMS inbred are male sterile as a result of factors resulting fromthe cytoplasmic, as opposed to the nuclear, genome. Thus, thischaracteristic is inherited exclusively through the female parent incorn plants, since only the female provides cytoplasm to the fertilizedseed. CMS plants are fertilized with pollen from another inbred that isnot male-sterile. Pollen from the second inbred may or may notcontribute genes that make the hybrid plants male-fertile. Seed fromdetasseled fertile corn and CMS produced seed of the same hybrid can beblended to insure that adequate pollen loads are available forfertilization when the hybrid plants are grown.

[0019] There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 to Brar et al. and chromosomal translocations asdescribed by Patterson in U.S. Pat. Nos. 3,861,709 and 3,710,511. Theseand all patents referred to are incorporated by reference. In additionto these methods, Albertsen et al., U.S. Pat. No. 5,432,068 havedeveloped a system of nuclear male sterility which includes: identifyinga gene which is critical to male fertility, silencing this native genewhich is critical to male fertility; removing the native promoter fromthe essential male fertility gene and replacing it with an induciblepromoter; inserting this genetically engineered gene back into theplant; and thus creating a plant that is male sterile because theinducible promoter is not “on” resulting in the male fertility gene notbeing transcribed. Fertility is restored by inducing, or turning “on”,the promoter, which in turn allows the gene that confers male fertilityto be transcribed.

[0020] There are many other methods of conferring genetic male sterilityin the art, each with its own benefits and drawbacks. These methods usea variety of approaches such as delivering into the plant a geneencoding a cytotoxic substance associated with a male tissue specificpromoter or an anti-sense system in which a gene critical to fertilityis identified and an antisense to that gene is inserted in the plant(see, Fabinjanski, et al. EPO 89/3010153.8 publication no. 329, 308 andPCT application PCT/CA90/00037 published as WO 90/08828).

[0021] Another version useful in controlling male sterility makes use ofgametocides. Gametocides are not a genetic system, but rather a topicalapplication of chemicals. These chemicals affect cells that are criticalto male fertility. The application of these chemicals affects fertilityin the plants only for the growing season in which the gametocide isapplied (see Carlson, G. R., U.S. Pat. No. 4,936,904). Application ofthe gametocide, timing of the application and genotype specificallyoften limit the usefulness of the approach.

[0022] Corn is an important and valuable field crop. Thus, a continuinggoal of plant breeders is to develop stable, high yielding corn hybridsthat are agronomically sound. The reasons for this goal are obviously tomaximize the amount of grain produced on the land used and to supplyfood for both animals and humans. To accomplish this goal, the cornbreeder must select and develop corn plants that have the traits thatresult in superior parental lines for producing hybrids.

SUMMARY OF THE INVENTION

[0023] According to the invention, there is provided a novel inbred cornline, designated HX894. This invention thus relates to the seeds ofinbred corn line HX894, to the plants of inbred corn line HX894 and tomethods for producing a corn plant produced by crossing the inbred lineHX894 with itself or another corn line, and to methods for producing acorn plant containing in its genetic material one or more transgenes andto the transgenic corn plants produced by that method. This inventionalso relates to methods for producing other inbred corn lines derivedfrom inbred corn line HX894 and to the inbred corn lines derived by theuse of those methods. This invention further relates to hybrid cornseeds and plants produced by crossing the inbred line HX894 with anothercorn line.

[0024] The inbred corn plant of the invention may further comprise, orhave, a cytoplasmic factor that is capable of conferring male sterility.Parts of the corn plant of the present invention are also provided, suchas e.g., pollen obtained from an inbred plant and an ovule of the inbredplant.

[0025] In another aspect, the present invention provides regenerablecells for use in tissue culture or inbred corn plant HX894. The tissueculture will preferably be capable of regenerating plants having thephysiological and morphological characteristics of the foregoing inbredcorn plant, and of regenerating plants having substantially the samegenotype as the foregoing inbred corn plant. Preferably, the regenerablecells in such tissue cultures will be embryos, protoplasts, meristematiccells, callus, pollen, leaves, anthers, roots, root tips, silk, flowers,kernels, ears, cobs, husks or stalks. Still further, the presentinvention provides corn plants regenerated from the tissue cultures ofthe invention.

Definitions

[0026] In the description and tables which follow, a number of terms areused. In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

[0027] Predicted RM. This trait for a hybrid, predicted relativematurity (RM), is based on the harvest moisture of the grain. Therelative maturity rating is based on a known set of checks and utilizesconventional maturity systems such as the Minnesota Relative MaturityRating System.

[0028] MN RM. This represents the Minnesota Relative Maturity Rating (MNRM) for the hybrid and is based on the harvest moisture of the grainrelative to a standard set of checks of previously determined MN RMrating. Regression analysis is used to compute this rating.

[0029] Yield (Bushels/Acre). The yield in bushels/acre is the actualyield of the grain at harvest adjusted to 15.5% moisture.

[0030] Moisture. The moisture is the actual percentage moisture of thegrain at harvest.

[0031] GDU Silk. The GDU silk (=heat unit silk) is the number of growingdegree units (GDU) or heat units required for an inbred line or hybridto reach silk emergence from the time of planting. Growing degree unitsare calculated by the Barger Method, where the heat units for a 24-hourperiod are: ${GDU} = {\frac{\left( {{Max}.{+ {Min}}} \right)}{2} - 50.}$

[0032] The highest maximum used is 86° F. and the lowest minimum used is50° F. For each hybrid, it takes a certain number of GDUs to reachvarious stages of plant development. GDUs are a way of measuring plantmaturity.

[0033] Stalk Lodging. This is the percentage of plants that stalk lodge,i.e., stalk breakage, as measured by either natural lodging or pushingthe stalks determining the percentage of plants that break off below theear. This is a relative rating of a hybrid to other hybrids forstandability.

[0034] Root Lodging. The root lodging is the percentage of plants thatroot lodge; i.e., those that lean from the vertical axis at anapproximate 300 angle or greater would be counted as root lodged.

[0035] Plant Height. This is a measure of the height of the hybrid fromthe ground to the tip of the tassel, and is measured in centimeters.

[0036] Ear Height. The ear height is a measure from the ground to theear node attachment, and is measured in centimeters.

[0037] Dropped Ears. This is a measure of the number of dropped ears perplot, and represents the percentage of plants that dropped an ear priorto harvest.

[0038] Allele. The allele is any of one or more alternative forms of agene, all of which alleles relate to one trait or characteristic. In adiploid cell or organism, the two alleles of a given gene occupycorresponding loci on a pair of homologous chromosomes.

[0039] Backcrossing. Backcrossing is a process in which a breederrepeatedly crosses hybrid progeny back to one of the parents, forexample, a first generation hybrid F₁ with one of the parental genotypesof the F₁ hybrid.

[0040] Quantitative Trait Loci (QTL). Quantitative trait loci (QTL)refer to genetic loci that control to some degree numericallyrepresentable traits that are usually continuously distributed.

[0041] Regeneration. Regeneration refers to the development of a plantfrom tissue culture.

[0042] Single Gene Converted. Single gene converted or conversion plantrefers to plants which are developed by a plant breeding techniquecalled backcrossing wherein essentially all of the desired morphologicaland physiological characteristics of an inbred are recovered in additionto the single gene transferred into the inbred via the backcrossingtechnique or via genetic engineering.

DETAILED DESCRIPTION OF THE INVENTION

[0043] Inbred corn line HX894 is a yellow dent corn with superiorcharacteristics, and provides an excellent parental line in crosses forproducing first generation (F₁) hybrid corn.

[0044] Standard pedigree ear-row selection method was used. Selfing andselection were conducted for seven generations in the development ofHX894. The selection criteria used during the development of HX894 were:grain yield; high plant density tolerance, good stand establishment,silking and pollen shedding ability, stalk and root strength, stay greenappearance during senescence, seed quality and disease tolerance.

[0045] Inbred corn line HX894 has the following morphologic and othercharacteristics (based primarily on data collected at Hooper, Nebr.)

Variety Description Information

[0046] VARIETY DESCRIPTION INFORMATION TYPE: Dent REGION WHEREDEVELOPED: Northcentral U.S. MATURITY: Days Heat Units Days to flower:81 1515 ${{Heat}\quad {Units}}:={\frac{\begin{matrix}\left\lbrack {{{Max}.\quad {Temp}.\quad \left( {\leq {86{^\circ}\quad {F.}}} \right)} +} \right. \\\left. {{Min}.\quad {Temp}.\quad \left( {\geq {50{^\circ}\quad {F.}}} \right)} \right\rbrack\end{matrix}}{2} - 50}$

PLANT: Plant Height: 71.8 inches Ear Height: 28.2 inches LEAF: Width:8.1 cm Length: 76.6 cm Number of leaves: 16.8 Leaf Sheath Pubescence:Moderate Marginal Waves: Some TASSEL: Number of Branches: 7.8 BranchAngle: 60-80° Tassel Length: 65 cm Anther Color: Pink EAR: Silk Color:Red Ear Length: 12.5-14.0 cm Number of Kernel Rows: 14-16 Shank Length:6.0 cm COB: Cob Color: Red

[0047] This invention is also directed to methods for producing a cornplant by crossing a first parent corn plant with a second parent cornplant, wherein the first or second corn plant is the inbred corn plantfrom the line HX894. Further, both first and second parent corn plantsmay be from the inbred line HX894. Therefore, any methods using theinbred corn line HX894 are part of this invention: selfing, backcrosses,hybrid breeding, and crosses to populations. Any plants produced usinginbred corn line HX894 as a parent are within the scope of thisinvention. Advantageously, the inbred corn line is used in crosses withother corn varieties to produce first generation (F₁) corn hybrid seedand plants with superior characteristics.

[0048] Some of the criteria used to select ears in various generationsinclude: yield, stalk quality, root quality, disease tolerance, lateplant greenness, late season plant intactness, ear retention, pollenshedding ability and silking ability. During the development of theline, crosses were made to inbred testers for the purpose of estimatingthe line's general and specific combining ability, and evaluations wererun by the Waterloo Research Station. The inbred was evaluated furtheras a line and in numerous crosses by other research stations across theCorn Belt. The inbred has proven to have a very good combining abilityin hybrid combinations.

[0049] The inbred has shown uniformity and stability within the limitsof environmental influence for the traits. It has been self-pollinatedand ear-rowed a sufficient number of generations, with careful attentionto uniformity of plant type to ensure homozygosity and phenotypicstability necessary to use in commercial production. The line has beenincreased both by hand and sibbed in isolated fields with continuedobservations for uniformity. No variant traits have been observed or areexpected in HX894.

Further Embodiments of the Invention

[0050] This invention also is directed to methods for producing a cornplant by crossing a first parent corn plant with a second parent cornplant wherein either the first or second parent corn plant is an inbredcorn plant of the line HX894. Further, both first and second parent cornplants can come from the inbred corn line HX894. Still further, thisinvention also is directed to methods for producing an inbred corn lineHX894-derived corn plant by crossing inbred corn line HX894 with asecond corn plant and growing the progeny seed, and repeating thecrossing and growing steps with the inbred corn line HX894-derived plantfrom 0 to 7 times. Thus, any such methods using the inbred corn lineHX894 are part of this invention: selfing, backcrosses, hybridproduction, crosses to populations, and the like. All plants producedusing inbred corn line HX894 as a parent are within the scope of thisinvention, including plants derived from inbred corn line HX894.Advantageously, the inbred corn line is used in crosses with other,different, corn inbreds to produce first generation (F₁) corn hybridseeds and plants with superior characteristics.

[0051] It should be understood that the inbred can, through routinemanipulation of cytoplasmic or other factors, be produced in amale-sterile form. Such embodiments are also contemplated within thescope of the present claims.

[0052] As used herein, the term plant includes plant cells, plantprotoplasts, plant cell tissue cultures from which corn plants can beregenerated, plant calli, plant clumps and plant cells that are intactin plants or parts of plants, such as embryos, pollen, ovules, flowers,kernels, ears, cobs, leaves, husks, stalks, roots, root tips, anthers,silk and the like.

[0053] Duncan, et al., Planta 165:322-332 (1985) reflects that 97% ofthe plants cultured that produced callus were capable of plantregeneration. Subsequent experiments with both inbreds and hybridsproduced 91% regenerable callus that produced plants. In a further studyin 1988, Songstad, et al., Plant Cell Reports 7:262-265 (1988), reportsseveral media additions that enhance regenerability of callus of twoinbred lines. Other published reports also indicated that“nontraditional” tissues are capable of producing somatic embryogenesisand plant regeneration. K. P. Rao et al., Maize Genetics CooperationNewsletter, 60:64-65 (1986), refers to somatic embryogenesis from glumecallus cultures and B. V. Conger, et al., Plant Cell Reports, 6:345-347(1987) indicates somatic embryogenesis from the tissue cultures of cornleaf segments. Thus, it is clear from the literature that the state ofthe art is such that these methods of obtaining plants are, and were,“conventional” in the sense that they are routinely used and have a veryhigh rate of success.

[0054] Tissue culture of corn is described in European PatentApplication, publication 160,390, incorporated herein by reference. Corntissue culture procedures are also described in Green and Rhodes, “PlantRegeneration in Tissue Culture of Maize,” Maize for Biological Research(Plant Molecular Biology Association, Charlottesville, Va. 367-372,(1982)) and in Duncan et al., “The Production of Callus Capable of PlantRegeneration from Immature Embryos of Numerous Zea Mays Genotypes,” 165Planta 322:332 (1985). Thus, another aspect of this invention is toprovide cells which upon growth and differentiation produce corn plantshaving the physiological and morphological characteristics of inbredcorn line HX894.

[0055] The utility of inbred corn line HX894 also extends to crosseswith other species. Commonly, suitable species will be of the familyGraminaceae, and especially of the genera Zea, Tripsacum, Croix,Schlerachne, Polytoca, Chionachne, and Trilobachne, of the tribeMaydeae. Potentially suitable for crosses with HX894 may be the variousvarieties of grain sorghum, Sorghum bicolor (L.) Moench.

[0056] With the advent of molecular biological techniques that haveallowed the isolation and characterization of genes that encode specificprotein products, scientists in the field of plant biology developed astrong interest in engineering the genome of plants to contain andexpress foreign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively as“transgenes”. Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transformedversions of the claimed inbred line.

[0057] Plant transformation involves the construction of an expressionvector which will function in plant cells. Such a vector comprises DNAcomprising a gene under control of or operatively linked to a regulatoryelement (for example, a promoter). The expression vector may contain oneor more such operably linked gene/regulatory element combinations. Thevector(s) may be in the form of a plasmid, and can be used alone or incombination with other plasmids, to provide transformed corn plants,using transformation methods as described below to incorporatetransgenes into the genetic material of the corn plant(s).

[0058] Expression Vectors for Corn Transformation

[0059] Marker Genes—Expression vectors include at least one geneticmarker, operably linked to a regulatory element (a promoter, forexample) that allows transformed cells containing the marker to beeither recovered by negative selection, i.e., inhibiting growth of cellsthat do not contain the selectable marker gene, or by positiveselection, i.e., screening for the product encoded by the geneticmarker. Many commonly used selectable marker genes for planttransformation are well known in the transformation arts, and include,for example, genes that code for enzymes that metabolically detoxify aselective chemical agent which may be an antibiotic or a herbicide, orgenes that encode an altered target which is insensitive to theinhibitor. A few positive selection methods are also known in the art.

[0060] One commonly used selectable marker gene for plant transformationis the neomycin phosphotransferase II (nptII) gene, isolated fromtransposon Tn5, which when placed under the control of plant regulatorysignals confers resistance to kanamycin. Fraley et al., Proc. Natl.Acad. Sci. U.S.A., 80:4803 (1983). Another commonly used selectablemarker gene is the hygromycin phosphotransferase gene which confersresistance to the antibiotic hygromycin. Vanden Elzen et al., Plant Mol.Biol., 5:299 (1985).

[0061] Additional selectable marker genes of bacterial origin thatconfer resistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase,the bleomycin resistance determinant. Hayford et al., Plant Physiol.86:1216 (1988), Jones et al., Mol. Gen. Genet., 210:86 (1987), Svab etal., Plant Mol. Biol. 14:197 (1990<Hille et al., Plant Mol. Biol. 7:171(1986). Other selectable marker genes confer resistance to herbicidessuch as glyphosate, glufosinate or broxynil. Comai et al., Nature317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618 (1990) andStalker et al., Science 242:419-423 (1988).

[0062] Other selectable marker genes for plant transformation are not ofbacterial origin. These genes include, for example, mouse dihydrofolatereductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plantacetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet. 13:67(1987), Shah et al., Science 233:478 (1986), Charest et al., Plant CellRep. 8:643 (1990).

[0063] Another class of marker genes for plant transformation requirescreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include β-glucuronidase (GUS,β-galactosidase, luciferase and chloramphenicol, acetyltransferase.Jefferson, R. A., Plant Mol. Biol. Rep. 5:387 (1987), Teeri et al., EMBOJ. 8:343 (1989), Koncz et al., Proc. Natl. Acad. Sci U.S.A. 84:131(1987), DeBlock et al., EMBO J. 3:1681 (1984). Another approach to theidentification of relatively rare transformation events has been use ofa gene that encodes a dominant constitutive regulator of the Zea maysanthocyanin pigmentation pathway. Ludwig et al., Science 247:449 (1990).

[0064] Recently, in vivo methods for visualizing GUS activity that donot require destruction of plant tissue have been made available.Molecular Probes publication 2908, Imagene Green™, p. 1-4 (1993) andNaleway et al., J. Cell Biol. 115:151a (1991). However, these in vivomethods for visualizing GUS activity have not proven useful for recoveryof transformed cells because of low sensitivity, high fluorescentbackgrounds and limitations associated with the use of luciferase genesas selectable markers.

[0065] More recently, a gene encoding Green Fluorescent Protein (GFP)has been utilized as a marker for gene expression in prokaryotic andeukaryotic cells. Chalfie et al., Science 263:802 (1994). GFP andmutants of GFP may be used as screenable markers.

[0066] Promoters—Genes included in expression vectors must be driven bynucleotide sequence comprising a regulatory element, for example, apromoter. Several types of promoters are now well known in thetransformation arts, as are other regulatory elements that can be usedalone or in combination with promoters.

[0067] As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred”.Promoters which initiate transcription only in certain tissue arereferred to as “tissue-specific”. A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter which is active under mostenvironmental conditions.

[0068] Inducible Promoters—An inducible promoter is operably linked to agene for expression in corn. Optionally, the inducible promoter isoperably linked to a nucleotide sequence encoding a signal sequencewhich is operably linked to a gene for expression in corn. With aninducible promoter the rate of transcription increases in response to aninducing agent.

[0069] Any inducible promoter can be used in the instant invention. SeeWard et al., Plant Mol. Biol. 22:361-366 (1993). Exemplary induciblepromoters include, but are not limited to, that from the ACEI systemwhich responds to copper (Meft et al., PNAS 90:4567-4571 (1993)); In2gene from maize which responds to benzenesulfonamide herbicide safeners(Hershey et al., Mol. Gen Genetics 227:229-237 (1991) and Gatz et al.,Mol. Gen. Genetics 243:32-38 (1994)) or Tet repressor from Tn10 (Gatz etal., Mol. Gen. Genetics 227:229-237 (1991). A particularly preferredinducible promoter is a promoter that responds to an inducing agent towhich plants do not normally respond. An exemplary inducible promoter isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone. Schena etal., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991).

[0070] Constitutive Promoters—A constitutive promoter is operably linkedto a gene for expression in corn or the constitutive promoter isoperably linked to a nucleotide sequence encoding a signal sequencewhich is operably linked to a gene for expression in corn.

[0071] Many different constitutive promoters can be utilized in theinstant invention. Exemplary constitutive promoters include, but are notlimited to, the promoters from plant viruses such as the 35S promoterfrom CaMV (Odell et al., Nature 313:810-812 (1985) and the promotersfrom such genes as rice actin (McElroy et al., Plant Cell 2:163-171(1990)); ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632(1989) and Christensen et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU(Last et al., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten etal., EMBO J. 3:2723-2730 (1984)) and maize H3 histone (Lepetit et al.,Mol. Gen. Genetics 231:276-285 (1992) and Atanassova et al., PlantJournal 2 (3): 291-300 (1992)).

[0072] The ALS promoter, Xba1/NcoI fragment 5′ to the Brassica napusALS3 structural gene (or a nucleotide sequence similarity to saidXba1/NcoI fragment), represents a particularly useful constitutivepromoter. See PCT application WO96/30530.

[0073] Tissue-specific or Tissue-preferred Promoters—A tissue-specificpromoter is operably linked to a gene for expression in corn.Optionally, the tissue-specific promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in corn. Plants transformed with a gene ofinterest operably linked to a tissue-specific promoter produce theprotein product of the transgene exclusively, or preferentially, in aspecific tissue.

[0074] Any tissue-specific or tissue-preferred promoter can be utilizedin the instant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,such as that from the phaseolin gene (Murai et al., Science 23:476-482(1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. U.S.A.82:3320-3324 (1985)); a leaf-specific and light-induced promoter such asthat from cab or rubisco (Simpson et al., EMBO J. 4(11):2723-2729 (1985)and Timko et al., Nature 318:579-582 (1985)); an anther-specificpromoter such as that from LAT52 (Twell et al., Mol. Gen. Genetics217:240-245 (1989)); a pollen-specific promoter such as that from Zm13(Guerrero et al., Mol. Gen. Genetics 244:161-168 (1993)) or amicrospore-preferred promoter such as that from apg (Twell et al., Sex.Plant Reprod. 6:217-224 (1993).

[0075] Signal Sequences for Targeting Proteins to SubcellularCompartments—Transport of protein produced by transgenes to asubcellular compartment such as the chloroplast, vacuole, peroxisome,glyoxysome, cell wall or mitochondroin or for secretion into theapoplast, is accomplished by means of operably linking the nucleotidesequence encoding a signal sequence to the 5′ and/or 3′ region of a geneencoding the protein of interest. Targeting sequences at the 5′ and/or3′ end of the structural gene may determine, during protein synthesisand processing, where the encoded protein is ultimatelycompartmentalized.

[0076] The presence of a signal sequence directs a polypeptide to eitheran intracellular organelle or subcellular compartment or for secretionto the apoplast. Many signal sequences are known in the art. See, forexample Becker et al., Plant Mol. Biol. 20:49 (1992), Close, P. S.,Master's Thesis, Iowa State University (1993), Knox, C., et al.,“Structure and Organization of Two Divergent Alpha-Amylase Genes fromBarley”, Plant Mol. Biol. 9:3-17 (1987), Lerner et al., Plant Physiol.91:124-129 (1989), Fontes et al., Plant Cell 3:483-496 (1991), Matsuokaet al., Proc. Natl. Acad. Sci. 88:834 (1991), Gould et al., J. Cell.Biol. 108:1657 (1989), Creissen et al., Plant J. 2:129 (1991), Kalderon,et al., A short amino acid sequence able to specify nuclear location,Cell 39:499-509 (1984), Steifel, et al., Expression of a maize cell wallhydroxyproline-rich glycoprotein gene in early leaf and root vasculardifferentiation, Plant Cell 2:785-793 (1990).

[0077] Foreign Protein Genes and Agronomic Genes—With transgenic plantsaccording to the present invention, a foreign protein can be produced incommercial quantities. Thus, techniques for the selection andpropagation of transformed plants, which are well understood in the art,yield a plurality of transgenic plants which are harvested in aconventional manner, and a foreign protein then can be extracted from atissue of interest or from total biomass. Protein extraction from plantbiomass can be accomplished by known methods which are discussed, forexample, by Heney and Orr, Anal. Biochem. 114:92-6 (1981).

[0078] According to a preferred embodiment, the transgenic plantprovided for commercial production of foreign protein is corn. Inanother preferred embodiment, the biomass of interest is seed. For therelatively small number of transgenic plants that show higher levels ofexpression, a genetic map can be generated, primarily via conventionalRFLP, PCR and SSR analysis, which identifies the approximate chromosomallocation of the integrated DNA molecule. For exemplary methodologies inthis regard, see Glick and Thompson, Methods in Plant Molecular Biologyand Biotechnology CRC Press, Boca Raton 269:284 (1993). Map informationconcerning chromosomal location is useful for proprietary protection ofa subject transgenic plant. If unauthorized propagation is undertakenand crosses made with other germplasm, the map of the integration regioncan be compared to similar maps for suspect plants, to determine if thelatter have a common parentage with the subject plant. Map comparisonswould involve hybridizations, RFLP, PCR, SSR and sequencing, all ofwhich are conventional techniques.

[0079] Likewise, by means of the present invention, agronomic genes canbe expressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

[0080] Genes That Confer Resistance to Pests or Disease and That Encode:

[0081] A. Plant disease resistance genes. Plant defenses are oftenactivated by specific interaction between the product of a diseaseresistance gene (R) in the plant and the product of a correspondingavirulence (Avr) gene in the pathogen. A plant inbred line can betransformed with cloned resistance gene to engineer plants that areresistant to specific pathogen strains. See, for example Jones et al.,Science 266:789 (1994) (cloning of the tomato Cf-9 gene for resistanceto Cladosporium fulvum); Martin et al., Science 262:1432 (1993) (tomatoPto gene for resistance to Pseudomonas syringae pv. Tomato encodes aprotein kinase); Mindrinos et al., Cell 78:1089 (1994) (Arabidopsis RSP2gene for resistance to Pseudomonas syringae).

[0082] B. A Bacillus thuringiensis protein, a derivative thereof or asynthetic polypeptide modeled thereon. See, for example, Geiser et al.,Gene 48:109 (1986), who disclose the cloning and nucleotide sequence ofa Bt δ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxingenes can be purchased from American Type Culture Collection, Manassas,Va., for example, under ATCC Accession Nos. 40098, 67136, 31995 and31998.

[0083] C. A lectin. See, for example, the disclose by Van Damme et al.,Plant Molec. Biol. 24:25 (1994), who disclose the nucleotide sequencesof several Clivia miniata mannose-binding lectin genes.

[0084] D. A vitamin-binding protein such as avidin. See PCT applicationUS93/06487, the contents of which are hereby incorporated by reference.The application teaches the use of avidin and avidin homologues aslarvicides against insect pests.

[0085] E. An enzyme inhibitor, for example, a protease or proteinaseinhibitor or an amylase inhibitor. See, for example, Abe et al., J.Biol. Chem. 262:16793 (1987) (nucleotide sequence of rice cysteineproteinase inhibitor), Huub et al., Plant Molec. Biol. 21:985 (1993)(nucleotide sequence of cDNA encoding tobacco proteinase inhibitor 1),Sumitani et al., Biosci. Biotech. Biochem. 57:1243 (1993) (nucleotidesequence of Streptomyces nitrosporeus α-amylase inhibitor).

[0086] F. An insect-specific hormone or pheromone such as an ecdysteroidand juvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

[0087] G. An insect-specific peptide or neuropeptide which, uponexpression, disrupts the physiology of the affected pest. For example,see the disclosures of Regan, J. Biol. Chem. 269:9 (1994) (expressioncloning yields DNA coding for insect diuretic hormone receptor), andPratt et al., Biochem. Biophys. Res. Comm. 163:1243 (1989) (anallostatin is identified in Diploptera puntata). See also U.S. Pat. No.5,266,317 to Tomalski et al., who disclose genes encodinginsect-specific, paralytic neurotoxins.

[0088] H. An insect-specific venom produced in nature by a snake, awasp, etc. For example, see Pang et al., Gene 116:165 (1992), fordisclosure of heterologous expression in plants of a gene coding for ascorpion insectotoxic peptide.

[0089] I. An enzyme responsible for a hyper accumulation of amonterpene, a sesquiterpene, a steroid, hydroxamic acid, aphenylpropanoid derivative or another non-protein molecule withinsecticidal activity.

[0090] J. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scoff et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence ofthe parsley ubi4-2 polyubiquitin gene.

[0091] K. A molecule that stimulates signal transduction. For example,see the disclosure by Botella et al., Plant Molec. Biol. 24:757 (1994),of nucleotide sequences for mung bean calmodulin cDNA clones, and Griesset al., Plant Physiol. 104:1467 (1994), who provide the nucleotidesequence of a maize calmodulin cDNA clone.

[0092] L. A hydrophobic moment peptide. See PCT application WO95/16776(disclosure of peptide derivatives of Tachyplesin which inhibit fungalplant pathogens) and PCT application WO95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference.

[0093] M. A membrane permease, a channel former or a channel blocker.For example, see the disclosure of Jaynes et al., Plant Sci 89:43(1993), of heterologous expression of a cecropin-β, lytic peptide analogto render transgenic tobacco plants resistant to Pseudomonassolanacearum.

[0094] N. A viral-invasive protein or a complex toxin derived therefrom.For example, the accumulation of viral coat proteins in transformedplant cells imparts resistance to viral infection and/or diseasedevelopment effected by the virus from which the coat protein gene isderived, as well as by related viruses. See Beachy et al., Ann. rev.Phytopathol. 28:451 (1990). Coat protein-mediated resistance has beenconferred upon transformed plants against alfalfa mosaic virus, cucumbermosaic virus, tobacco streak virus, potato virus X, potato virus Y,tobacco etch virus, tobacco rattle virus and tobacco mosaic virus. Id.

[0095] O. An insect-specific antibody or an immunotoxin derivedtherefrom. Thus, an antibody targeted to a critical metabolic functionin the insect gut would inactivate an affected enzyme, killing theinsect. Cf. Taylor et al., Abstract #497, Seventh Int'l Symposium onMolecular Plant-Microbe Interactions (Edinburgh, Scotland) (1994)(enzymatic inactivation in transgenic tobacco via production ofsingle-chain antibody fragments).

[0096] P. A virus-specific antibody. See, for example, Tavladoraki etal., Nature 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

[0097] Q. A developmental-arrestive protein produced in nature by apathogen or a parasite. Thus, fungal endo α-1,4-D-polygalacturonasesfacilitate fungal colonization and plant nutrient release bysolubilizing plant cell wall homo-α-1,4-D-galacturonase. See Lamb etal., Bio/Technology 10:1436 (1992). The cloning and characterization ofa gene which encodes a bean endopolygalacturonase-inhibiting protein isdescribed by Toubart et al., Plant J. 2:367 (1992).

[0098] R. A development-arrestive protein produced in nature by a plant.For example, Logemann et al., Bioi/Technology 10:305 (1992), have shownthat transgenic plants expressing the barley ribosome-inactivating genehave an increased resistance to fungal disease.

[0099] Genes That Confer Resistance to a Herbicide, For Example:

[0100] A. A herbicide that inhibits the growing point or meristem, suchas an imidazalinone or a sulfonylurea. Exemplary genes in this categorycode for mutant ALS and AHAS enzyme as described, for example, by Lee etal., EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet. 80:449(1990), respectively.

[0101] B. Glyphosate (resistance impaired by mutant5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase, PAT and Streptomyces hygroscopicusphosphinothricin-acetyl transferase, bar, genes), and pyridinoxy orphenoxy propionic acids and cycloshexones (ACCase inhibitor-encodinggenes). See, for example, U.S. Pat. No. 4,940,835 to Shah, et al., whichdiscloses the nucleotide sequence of a form of EPSP which can conferglyphosate resistance. A DNA molecule encoding a mutant aroA gene can beobtained under ATCC accession number 39256, and the nucleotide sequenceof the mutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai.European patent application No. 0 333 033 to Kumada et al., and U.S.Pat. No. 4,975,374 to Goodman et al., disclose nucleotide sequences ofglutamine synthetase genes which confer resistance to herbicides such asL-phosphinothricin. The nucleotide sequence of aphosphinothricin-acetyl-transferase gene is provided in Europeanapplication No. 0 242 246 to Leemans et al., DeGreef et al.,Bio/Technology 7:61 (1989), describe the production of transgenic plantsthat express chimeric bar genes coding for phosphinothricin acetyltransferase activity. Exemplary of genes conferring resistance tophenoxy propionic acids and cycloshexones, such as sethoxydim andhaloxyfop are the Acc1-S1, Acc1-S2 and Acc1-S3 genes described byMarshall et al., Theor. Appl. Genet. 83:435 (1992).

[0102] C. A herbicide that inhibits photosynthesis, such as a triazine(psbA and gs+genes) and a benzonitrile (nitrilase gene). Przibilla etal., Plant Cell 3:169 (1991), describe the transformation ofChlamydomonas with plasmids encoding mutant psbA genes. Nucleotidesequences for nitrilase genes are disclosed in U.S. Pat. No. 4,810,648to Stalker, and DNA molecules containing these genes are available underATCC Accession Nos. 53435, 67441, and 67442. Cloning and expression ofDNA coding for a glutathione S-transferase is described by Hayes et al.,Biochem. J. 285:173 (1992).

[0103] Genes That Confer or Contribute to a Value-Added Trait, Such as:

[0104] Modified fatty acid metabolism, for example, by transforming aplant with an antisense gene of stearyl-ACP desaturase to increasestearic acid content of the plant. See Knultzon et al., Proc. Natl.Acad. Sci. U.S.A. 89:2624 (1992).

[0105] Decreased Phytate Content

[0106] 1) Introduction of a phytase-encoding gene would enhancebreakdown of phytate, adding more free phosphate to the transformedplant. For example, see Van Hartingsveldt et al., Gene 127:87 (1993),for a disclosure of the nucleotide sequence of an Aspergillus nigerphytase gene.

[0107] 2) A gene could be introduced that reduced phytate content. Inmaize, this, for example, could be accomplished, by cloning and thenreintroducing DNA associated with the single allele which is responsiblefor maize mutants characterized by low levels of phytic acid. See Raboyet al., Maydica 35:383 (1990).

[0108] Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza et al., J. Bacteol. 170:810(1988) (nucleotide sequence of Streptococcus mutantsfructosyltransferase gene), Steinmetz et al., Mol. Gen. Genet. 20:220(1985) (nucleotide sequence of Bacillus subtilis levansucrase gene), Penet al., Bio/Technology 10:292 (1992) (production of transgenic plantsthat express Bacillus licheniformis α-amylase), Elliot et al., PlantMolec. Biol. 21:515 (1993) (nucleotide sequences of tomato invertasegenes), Søogaard et al., J. Biol. Chem. 268:22480 (1993) (site-directedmutagenesis of barley α-amylase gene), and Fisher et al., Plant Physiol.102:1045 (1993) (maize endosperm starch branching enzyme 11).

[0109] Methods for Corn Transformation-Numerous methods for planttransformation have been developed, including biological and physical,plant transformation protocols. See, for example, Miki et al.,“Procedures for Introducing Foreign DNA into Plants” in Methods in PlantMolecular Biology and Biotechnology, Glick B. R. and Thompson, J. E.Eds. (CRC Press, Inc., Boca Raton, 1993) pages 67-88. In addition,expression vectors and in vitro culture methods for plant cell or tissuetransformation and regeneration of plants are available. See, forexample, Gruber et al., “Vectors for Plant Transformation” in Methods inPlant Molecular Biology and Biotechnology, Glick B. R. and Thompson, J.E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages 89-119.

[0110] Agrobacterium-mediated Transformation—One method for introducingan expression vector into plants is based on the natural transformationsystem of Agrobacterium. See, for example, Horsch et al., Science227:1229 (1985). A. tumefaciens and A. rhizogenes are plant pathogenicsoil bacteria which genetically transform plant cells. The Ti and Riplasmids of A. tumefaciens and A. rhizogenes, respectively, carry genesresponsible for genetic transformation of the plant. See, for example,Kado, C. I., Crit. Rev. Plant Sci. 10:1 (1991). Descriptions ofAgrobacterium vector systems and methods for Agrobacterium-mediated genetransfer are provided by Gruber et al., supra, Miki et al., supra, andMoloney et al., Plant Cell Reports 8:238 (1989). See also, U.S. Pat. No.5,591,616 issued Jan. 7, 1997.

[0111] Direct Gene Transfer—Despite the fact the host range forAgrobacterium-mediated transformation is broad, some major cereal cropspecies and gymnosperms have generally been recalcitrant to this mode ofgene transfer, even though some success has recently been achieved inrice and corn. Hiei et al., The Plant Journal 6:271-282 (1994) and U.S.Pat. No. 5,591,616 issued Jan. 7, 1997. Several methods of planttransformation, collectively referred to as direct gene transfer, havebeen developed as an alternative to Agrobacterium-mediatedtransformation.

[0112] A generally applicable method of plant transformation ismicroprojectile-mediated transformation wherein DNA is carried on thesurface of microprojectiles measuring 1 to 4 μm. The expression vectoris introduced into plant tissues with a biolistic device thataccelerates the microprojectiles to speeds of 300 to 600 m/s which issufficient to penetrate plant cell walls and membranes. Sanford et al.,Part. Sci. Technol. 5:27 (1987), Sanford, J. C., Trends Biotech. 6:299(1988), Klein et al., Bio/Technology 6:559-563 (1988), Sanford, J. C.,Physiol Plant 7:206 (1990), Klein et al., Biotechnology 10:268 (1992).In corn, several target tissues can be bombarded with DNA-coatedmicroprojectiles in order to produce transgenic plants, including, forexample, callus (Type I or Type II), immature embryos, and meristematictissue.

[0113] Another method for physical delivery of DNA to plants issonication of target cells. Zhang et al., Bio/Technology 9:996 (1991).Alternatively, liposome or spheroplast fusion have been used tointroduce expression vectors into plants. Deshayes et al., EMBO J.,4:2731 (1985), Christou et al., Proc Natl. Acad. Sci. U.S.A. 84:3962(1987). Direct uptake of DNA into protoplasts using CaCl₂ precipitation,polyvinyl alcohol or poly-L-omithine have also been reported. Hain etal., Mol. Gen. Genet. 199:161 (1985) and Draper et al., Plant CellPhysiol. 23:451 (1982). Electroporation of protoplasts and whole cellsand tissues have also been described. Donn et al., In Abstracts of VIIthInternational Congress on Plant Cell and Tissue Culture IAPTC, A2-38, p53 (1990); D'Halluin et al., Plant Cell 4:1495-1505 (1992) and Spenceret al., Plant Mol. Biol. 24:51-61 (1994).

[0114] Following transformation of corn target tissues, expression ofthe above-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

[0115] The foregoing methods for transformation would typically be usedfor producing a transgenic inbred line. The transgenic inbred line couldthen be crossed, with another (non-transformed or transformed) inbredline, in order to produce a new transgenic inbred line. Alternatively, agenetic trait which has been engineered into a particular corn lineusing the foregoing transformation techniques could be moved intoanother line using traditional backcrossing techniques that are wellknown in the plant breeding arts. For example, a backcrossing approachcould be used to move an engineered trait from a public, non-eliteinbred line into an elite inbred line, or from an inbred line containinga foreign gene in its genome into an inbred line or lines which do notcontain that gene. As used herein, “crossing” can refer to a simple X byY cross, or the process of backcrossing, depending on the context.

[0116] When the term inbred corn plant is used in the context of thepresent invention, this also includes any single gene conversions ofthat inbred. The term single gene converted plant as used herein refersto those corn plants which are developed by a plant breeding techniquecalled backcrossing wherein essentially all of the desired morphologicaland physiological characteristics of an inbred are recovered in additionto the single gene transferred into the inbred via the backcrossingtechnique. Backcrossing methods can be used with the present inventionto improve or introduce a characteristic into the inbred. The termbackcrossing as used herein refers to the repeated crossing of a hybridprogeny back to one of the parental corn plants for that inbred. Theparental corn plant which contributes the gene for the desiredcharacteristic is termed the nonrecurrent or donor parent. Thisterminology refers to the fact that the nonrecurrent parent is used onetime in the backcross protocol and therefore does not recur. Theparental corn plant to which the gene or genes from the nonrecurrentparent are transferred is known as the recurrent parent as it is usedfor several rounds in the backcrossing protocol (Poehiman & Sleper,1994; Fehr, 1987). In a typical backcross protocol, the original inbredof interest (recurrent parent) is crossed to a second inbred(nonrecurrent parent) that carries the single gene of interest to betransferred. The resulting progeny from this cross are then crossedagain to the recurrent parent and the process is repeated until a cornplant is obtained wherein essentially all of the desired morphologicaland physiological characteristics of the recurrent parent are recoveredin the converted plant, in addition to the single transferred gene fromthe nonrecurrent parent.

[0117] The selection of a suitable recurrent parent is an important stepfor a successful backcrossing procedure. The goal of a backcrossprotocol is to alter or substitute a single trait or characteristic inthe original inbred. To accomplish this, a single gene of the recurrentinbred is modified or substituted with the desired gene from thenonrecurrent parent, while retaining essentially all of the rest of thedesired genetic, and therefore the desired physiological andmorphological, constitution of the original inbred. The choice of theparticular nonrecurrent parent will depend on the purpose of thebackcross, one of the major purposes is to add some commerciallydesirable, agronomically important trait to the plant. The exactbackcrossing protocol will depend on the characteristic or trait beingaltered to determine an appropriate testing protocol. Althoughbackcrossing methods are simplified when the characteristic beingtransferred is a dominant allele, a recessive allele may also betransferred. In this instance it may be necessary to introduce a test ofthe progeny to determine if the desired characteristic has beensuccessfully transferred.

[0118] Many single gene traits have been identified that are notregularly selected for in the development of a new inbred but that canbe improved by backcrossing techniques. Single gene traits may or maynot be transgenic, examples of these traits include but are not limitedto, male sterility, waxy starch, herbicide resistance, resistance forbacterial, fungal, or viral disease, insect resistance, male fertility,enhanced nutritional quality, industrial usage, yield stability andyield enhancement. These genes are generally inherited through thenucleus. Some known exceptions to this are the genes for male sterility,some of which are inherited cytoplasmically, but still act as singlegene traits. Several of these single gene traits are described in U.S.Pat. Nos. 5,777,196; 5,948,957 and 5,969,212, the disclosures of whichare specifically hereby incorporated by reference.

[0119] Industrial Applicability—Corn is used as human food, livestockfeed, and as raw material in industry. The food uses of corn, inaddition to human consumption of corn kernels, include both products ofdry- and wet-milling industries. The principal products of corn drymilling are grits, meal and flour. The corn wet-milling industry canprovide corn starch, corn syrups, and dextrose for food use. Corn oil isrecovered from corn germ, which is a by-product of both dry- andwet-milling industries.

[0120] Corn, including both grain and non-grain portions of the plant,is also used extensively as livestock feed, primarily for beef cattle,dairy cattle, hogs and poultry.

[0121] Industrial uses of corn include production of ethanol, cornstarch in the wet-milling industry and corn flour in the dry-millingindustry. The industrial applications of corn starch and flour are basedon functional properties, such as viscosity, film formation, adhesiveproperties, and ability to suspend particles. The corn starch and flourhave application in the paper and textile industries. Other industrialuses include applications in adhesives, building materials, foundrybinders, laundry starches, explosives, oil-well muds and other miningapplications.

[0122] Plant parts other than the grain of corn are also used inindustry, for example: stalks and husks are made into paper andwallboard and cobs are used for fuel and to make charcoal.

[0123] The seed of inbred corn line HX894, the plant produced from theinbred seed, the hybrid corn plant produced from the crossing of theinbred, hybrid seed, and various parts of the hybrid corn plant andtransgenic versions of the foregoing, can be utilized for human food,livestock feed, and as a raw material in industry.

Tables

[0124] In the tables that follow, the traits and characteristics ofinbred corn line HX894 are given in hybrid combination. The datacollected on inbred corn line HX894 is presented for the keycharacteristics and traits. The tables present yield test informationabout HX894. HX894 was tested in several hybrid combinations at numerouslocations, with several replications per location. Information aboutthese hybrids, as compared to several check hybrids, is presented.

[0125] In the following tables, information for each hybrid includes:

[0126] 1. Hybrids are listed in column 1.

[0127] 2. The year of testing is in column 2.

[0128] 3. Mean yield, in bushels per acre, of the hybrid across alllocations is in column 3.

[0129] 4. A mean for the percentage moisture (% M) for the hybrid acrossall locations is in column 4.

[0130] 5. The Test Weight (TW) is a measure of the weight of the grainin pounds for a given volume (bushel) adjusted for 15.5 percentmoisture.

[0131] 6. A mean of the percentage of plants with root lodging (% Root)across all locations is in column 6.

[0132] 7. A mean of the percentage of plants with stalk lodging (%Stalk) across all locations is in column 7. TABLE 1 % % Hybrid YearYield % M TW Root Stalk HX894 × FR4901 2001 210.5 19.8 55.7 0.2 2.9LH244 × TR7322 2001 206.2 19.1 55.8 0.1 3.0 FR3912 × FR4901 2001 208.320.7 55.6 0.0 2.6 MBS3651 × SGI847 2001 203.7 20.0 55.1 0.0 3.6 No. ofComparisons 30 30 30 30 30

[0133] TABLE 2 % % Hybrid Year Yield % M TW Root Stalk HX894 × TR73222001 205.6 20.9 55.2 0.0 2.5 LH244 × TR7322 2001 206.2 19.1 55.8 0.1 3.0LH132 × SGI851 2001 200.8 20.0 55.3 0.0 3.3 No. of Comparisons 30 30 3030 30

Deposit Information

[0134] A deposit of the corn seed of this invention is maintained byHoegemeyer Hybrids, 1755 Hoegemeyer Road, Hooper, Nebr. 68031. Access tothis deposit will be available during the pendency of this applicationto persons determined by the Commissioner of Patents and Trademarks tobe entitled thereto under 37 CFR 1.14 and 35 USC 122. Upon allowance ofany claims in this application, all restrictions on the availability tothe public of the variety will be irrevocably removed by affordingaccess to a deposit of at least 2,500 seeds of the same variety with theAmerican Type Culture Collection, Manassas, Va.

[0135] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity andunderstanding. However, it will be obvious that certain changes andmodifications such as single gene modifications and mutations,somoclonal variants, variant individuals selected from large populationsof the plants of the instant inbred and the like may be practiced withinthe scope of the invention, as limited only by the scope of the appendedclaims.

What is claimed is:
 1. Seed of corn inbred line designated HX894,representative seed of said line having been deposited under ATCCAccession No. ______.
 2. A corn plant, or parts thereof, produced bygrowing the seed of claim
 1. 3. Pollen of the plant of claim
 2. 4. Anovule of the plant of claim
 2. 5. A method of producing a male sterilecorn plant comprising crossing the corn plant of claim 2 with a malesterile corn plant and harvesting the resultant seed.
 6. A tissueculture of regenerable cells from the corn plant of claim
 2. 7. A tissueculture according to claim 6, the cells or protoplasts of the tissueculture being from a tissue selected from the group consisting ofleaves, pollen, embryos, roots, root tips, anthers, silks, flowers,kernels, ears, cobs, husks, and stalks.
 8. A corn plant regenerated fromthe tissue culture of claim 6, wherein the regenerated plant is capableof expressing all the morphological and physiological characteristics ofinbred line HX894.
 9. A method for producing a hybrid corn seedcomprising crossing a first inbred parent corn plant with a secondinbred parent corn plant and harvesting the resultant hybrid corn seed,wherein said first inbred parent corn plant or second said parent cornplant is the corn plant of claim
 2. 10. A hybrid corn seed produced bythe method of claim
 9. 11. A hybrid corn plant, or parts thereof,produced by growing said hybrid corn seed of claim
 10. 12. A method ofproducing a corn seed by growing said hybrid corn plant of claim 11 andharvesting the resultant corn seed.
 13. A method for producing inbredHX894, representative seed of which have been deposited under ATCCAccession No. ______, comprising: a) planting a collection of seedcomprising seed of a hybrid, one of whose parents is inbred HX894, saidcollection also comprising seed of said inbred; b) growing plants fromsaid collection of seed; c) identifying inbred parent plants; d)controlling pollination in a manner which preserves the homozygosity ofsaid inbred parent plant; and e) harvesting the resultant seed.
 14. Theprocess of claim 13 wherein step (c) comprises identifying plants withdecreased vigor.
 15. A method for producing a HX894-derived corn plant,comprising: a) crossing inbred corn line HX894, representative seed ofsaid line having been deposited under ATCC accession number ______, witha second corn plant to yield progeny corn seed; b) growing said progenycorn seed, under plant growth conditions, to yield said HX894-derivedcorn plant; c) crossing said HX894-derived corn plant with itself oranother corn plant to yield additional HX894-derived progeny corn seed;d) growing said progeny corn seed of step (c) under plant growthconditions, to yield additional HX894-derived corn plants; and e)repeating the crossing and growing steps of (c) and (d) from 0 to 7times to generate further HX894-derived corn plants.
 16. A HX894-derivedcorn plant, or parts thereof, produced by the method of claim
 15. 17.The method of claim 15, still further comprising utilizing plant tissueculture methods to derive progeny of said HX894-derived corn plant. 18.A method for producing a corn plant that contains in its geneticmaterial one or more transgenes, comprising crossing the corn plant ofclaim 2 with either a second plant of another corn line, or atransformed corn plant of the line HX894, so that the genetic materialof the progeny that result from the cross contains the transgene(s)operably linked to a regulatory element.
 19. The method of claim 18,wherein said transgene is selected from the group consisting ofherbicide resistance and insect resistance.
 20. A method for developinga corn plant in a corn plant breeding program using plant breedingtechniques which include employing a corn plant, or its parts, as asource of plant breeding material comprising: using the corn plant, orits parts, of claim 2 as a source of said breeding material wherein saidplant breeding techniques are selected from the group consisting of:recurrent selection, backcrossing, pedigree breeding, restrictionfragment length polymorphism enhanced selection, genetic marker enhancedselection, and transformation.
 21. A corn plant, or parts thereof,produced by the method of claim 20.